Rheumatoid arthritis (RA), a chronic inflammatory disease of the joints, manifests as a chronic synovitis and progressive destruction of the joints, leukocyte infiltrates, and cartilage destruction and bone erosion. It is believed this destruction is supported and perpetuated by proinflammatory cytokines contributed by autoreactive T cells. Rodent models have been developed that mimic arthritis, and one such model, collagen-induced arthritis (CIA), requires immunization with heterologous collagen II. Oral tolerance and treatments with regulatory cytokines have been suggested as possible interventions to treat arthritis. One such new therapeutic is the regulatory cytokine, IL-35;however, its mode of action has yet to be determined. IL-35 is a heterodimer cytokine composed of IL-12p35 plus IL-27 EBI3 subunits. We recently expressed IL-35 as a single polypeptide using eukaryotic expression systems. The recombinant mouse IL-35 has the expected molecular weight, and it is recognized with antibodies to IL-12p35 and IL-27-EBI3. Functional analysis reveals that IL-35 can completely block development of clinical symptoms of CIA. This disease suppression does appear to be IL-10-dependent, produced by heterogeneous regulatory T cell subsets, including CD25+ CD4+ T cells, as well as CD39+ CD4+ T cells. Thus, additional studies are warranted to investigate the nature of these regulatory T cells and learn which mononuclear cells are responsive to IL-35's action. Given these findings, the hypothesis to be tested in this application is that IL-35 intervention will reduce disease severity and limit disease progression of CIA by the stimulation of regulatory T cells. To test this hypothesis, two Specific Aims are proposed. Studies in Specific Aim 1 will determine if IL-35 in a paracrine/autocrine fashion stimulates endogenous IL-35 production for treatment of CIA and determine the cell types involved to facilitate IL-35's action. Studies in Specific Aim 2 will define the regulatory T cell subset induced by IL-35 that is responsible for protection against CIA and determine which inflammatory T cell subset is diminished. These collective studies will provide the foundation of whether IL-35 eff can be considered in RA intervention strategies and whether alternative regulatory T cells can be defined that would facilitate IL-35's therapeutic impact in humans. PUBLIC HEALTH RELEVANCE: Herein this application we propose to evaluate the therapeutic potential of IL-35 to treat rheumatoid arthritis. The described studies will ascertain the ability and mechanisms used by IL-35 to treat autoimmune diseases such as arthritis. Once we understand its therapeutic potential, future studies can then determine optimal means to deliver IL-35 using viral or DNA vectors to treat or reduce arthritis.